The invention relates to a method for determining tissue content, such as lipid and protein content, in a tissue of a subject. The method involves modulating the temperature of the tissue being analyzed and collecting spectra emitted under differing temperature conditions.
There has long been considerable interest in the non-invasive monitoring of body chemistry and a need for reliable and precise quantitative methods for diagnosing medical abnormalities and for assessing the general condition of body tissues. While any approach that offers early and reliable warning of medical problems has some utility, noninvasive methods offer many advantages. Anticipation by a patient of pain and scarring associated with invasive procedures can cause delays in seeking medical attention. There is also a myriad of inconveniences, risks and difficulties associated with direct collection and contact with patient body fluids. For these reasons, there has been intense scientific and engineering research into devising noninvasive approaches to assessment and diagnosis of medical conditions.
Use of spectroscopic methods, while of considerable use in direct in vitro application to fluids, has not found equal in vivo application. In vivo sampling is substantially more complicated for a variety of reasons, although some of the challenges can be handled by reference to in vitro procedures. First, even in vitro procedures require at least some sample preparation before spectroscopic interrogation. But in vivo samples cannot be handled with nearly the ease of in vitro samples. All chemometric analyses benefit from the availability of samples having known composition of various analytes. Selectively modulated in vitro samples are much easier to synthesize or otherwise obtain than in vivo samples. Thus, samples for chemometric interpretation of in vivo samples can be expected to require specialized approaches to sample preparation and specifically designed methods for obtaining modulated samples of known composition. Long data collection times are needed to extract small signals from some samples, but in vivo sampling requires the patient to endure the waiting. Prolonged data collection is not always practical. Moreover, applying too much excitation light to in vivo samples can lead to catastrophic results.
Noninvasive in vivo chemical analysis of human and animal tissues has long been a goal of chemists and the medical community. Blood oximetry is an example of a noninvasive form of analysis that is now ubiquitous in intensive care and other situations. Noninvasive techniques involve contacting the tissue in question with some form of electromagnetic radiation, and detecting the effect of the contact on the radiation. The frequency range of the radiation and the choice of tissue to contact, determines the type of structural, concentration or other physico-chemical information available. Optimal application of noninvasive techniques for tissue analysis will require improved methods for isolating signals attributable to particular elements within tissues.
To overcome the limitations of the prior art, the invention provides a method for monitoring phase transitions, and thereby methods for determining lipid and protein content and identity in a tissue of a subject. The method can be performed in vivo and noninvasively. The method is preferably used with noninvasive spectroscopy, such as Raman spectroscopy, for the analysis of various features of tissue in a subject.
In one embodiment, the invention provides a method of determining lipid content of tissue in a subject. The method comprises contacting the tissue with electromagnetic radiation having an excitation wavelength, collecting the Raman spectra emitted by the tissue in a range of wavelengths associated with lipids, and altering the temperature of the tissue. Examples of a range of wavelengths associated with lipids include, but are not limited to, about 1450-1500 cmxe2x88x921 or about 2850-2890 cmxe2x88x921. The method further comprises repeating the contacting and collecting steps while the temperature of the tissue is altered, and analyzing the spectra collected to determine an amount of lipid present in the tissue. Preferably, the temperature of the tissue is altered by cooling. For example, the tissue can be cooled to about 1xc2x0 C. to about 35xc2x0 C. Preferably, the tissue is cooled to about 2xc2x0 C. to about 12xc2x0 C. In one embodiment, the analyzing comprises determining the difference in number of Raman shifted photons emitted by the tissue in the differing temperature conditions. Preferably, the tissue is a fingertip.
In another embodiment, the invention provides a method of determining protein content of tissue in a subject. The method comprises contacting the tissue with electromagnetic radiation having an excitation wavelength, collecting the Raman spectra emitted by the tissue in a range of wavelengths associated with protein, and altering the temperature of the tissue. Examples of a range of wavelengths associated with protein include, but are not limited to, about 1610-1700 cmxe2x88x921. The method further comprises repeating the contacting and collecting steps while the temperature of the tissue is altered, and analyzing the spectra collected to determine an amount of protein present in the tissue. Preferably, the temperature of the tissue is altered by cooling. For example, the tissue can be cooled by about 2 to about 35xc2x0 C. In one embodiment, the analyzing comprises determining the difference in number of Raman shifted photons emitted by the tissue in the differing temperature conditions. Preferably, the tissue is a fingertip.
In one embodiment, the method further comprises determining the depth of a source of the spectra emitted by the tissue. For example, the determining can comprise using a confocal lens system to collect emitted spectra. The analyzing can further comprise determining the type of lipid or protein present in the tissue based on the depth of the source of the spectra emitted by the tissue.
In a preferred embodiment, the method comprises applying a tissue modulation device of the invention to the tissue, passing current through the temperature regulating element so as to elevate or lower the temperature of the tissue, and passing electromagnetic radiation through the window of the device. Preferably, spectroscopic probing is performed when the temperature of the tissue has been elevated or lowered and when the temperature of the tissue is not elevated or lowered. The method can further comprise collecting Raman spectra emitted by the tissue. The collected spectra are then analyzed to determine the lipid content, lipid identity and/or protein content and identity of the tissue. Preferably, the analysis comprises determining the difference in number of Raman shifted photons emitted by the tissue at different temperatures.